Pillar[5]arene-Derived endo-Functionalized Molecular Tube for Mimicking Protein-Ligand Interactions

Articial tubular molecular pockets bearing polar functionalities on their inner surface are useful model systems for understanding the mechanisms of protein-ligand interactions in living systems. We herein report a pillar[5]arene-derived molecular tube, [P4-(OH)BPO], whose endo conformational isomer endo-[P4-(OH)BPO] possesses an inwardly pointing hydrogen-bond (H-bond) donor (OH) in its deep cavity, a strong H-bond acceptor (C=O) on the predominantly hydrophobic inner surface, rendering it a perfect protein binding pocket mimetic. By measuring the binding anity of this pocket-mimetic tube, we screened a library of various shape-complementary organic guests (1–38) resembling the fragment ligands in fragment-based drug design (FBDD). On the basis of the data for “fragment-pocket” complexes (1– 38) ⊂ endo-[P4-(OH)BPO], two rationally designed “lead molecules” (39 and 40) were identied to be able to enhance binding anity signicantly by forming H-bonds with both the donor and acceptor of endo-[P4-(OH)BPO]. The described work opens new avenues for developing pillar[n]arene-derived protein binding pocket-mimetic systems for studies on protein-ligand interactions and mechanisms of enzymatic reactions.


Introduction
Small molecules are the most common modality for new medicines, accounting for 67% of the FDAapproved drugs in 2019 1 . Almost all current small-molecule drugs act by targeting disease-related proteins in the human body and regulating their activity 2,3 . As the interaction between a protein and a small molecule is often through binding of the molecule in a pocket on the protein surface, characterization of such binding pockets is important to elucidate the disease mechanisms and design new drug molecules [4][5][6] . Along with various methods, model studies using biomimetic receptorsarti cially fabricated functional macrocyclic hosts -to mimic protein binding pockets are useful for understanding the nature of protein-ligand recognition 7,8 . During the past decades, the approach of fragment-based drug design (FBDD) has attracted increasing interest both in academia and pharmaceutical industry [9][10] . In FBDD, drug compounds were designed on the basis of protein-ligand bonding using fragment ligands with low complexity in chemical structures and low molecular weights [9][10] . It is well established that a protein binding pocket has an interior surface bearing both polar and nonpolar functionalities for synergistic hydrogen bonding and hydrophobic interactions with a guest molecule [4][5] . In a reported survey of fragment-pocket complexes, hydrogen bonds (H-bonds) stabilize 89% of the fragment-pocket complexes, with 74% of the entries displaying one to three H-bonds 10 . Therefore, in order to imitate the microenvironment within protein binding pockets, the interior surface of the biomimetic receptors must possess polar functionalities for H-bond formation. To this end, there have been reports on pocket-shaped receptors, such as cavitands 11 , glucose receptor 2 12 , aryl-extended calix [4]pyrrole receptors 13 , helical aromatic and oligoamide foldamers (Fig. 1a-e) 14,15 . Jiang's group recently made signi cant progress in transforming naphthalene-based tubular molecules into biomimetic tubes in which the inwardly directed functional groups improved their binding a nity and selectivity toward fragment ligands (Fig. 1f) [16][17][18] . As a class of tubular-shaped macrocyclic hosts, pillar[n]arenes [19][20][21] could seat various functional substituents on their rims, for instance, amino acids or short peptides, for mimicking transmembrane channels 22 . Nonetheless, all of these rim-embedded polar functional substituents pointed outwards, and there has been no report on mounting polar functionalities pointing inwardly to the cavity cores of pillar[n]arenes. Herein, we report the creation of a pillar [5]arene-derived tubular molecular pocket with an inward-pointing hydroxyl group, endo-[P4-(OH)BPO] (Fig. 1g), which could unidirectionally interact with various small shape-complementary polar molecular guests through synergistic hydrophobic and hydrogen bonding interactions within its tubular cavity, mimicking a fragment-pocket in FBDD or protein-ligand interaction event in a protein binding pocket.

Results And Discussion
Formation of endo-[P4-(OH)BPO]. Our group has previously developed unique methods to grow functional substituents on the rims of pillar[n]arenes 23 . We conjectured that a pillar[n]arene-derived protein binding pocket mimetic could be realized through mounting a polar functionality on the rim of a pillar[n]arene, and then forcing it to point towards the tubular core either through steric effect or substrate-induced conformational change. We therefore started our work from adding a small polar functionality, hydroxyl group, to the rim of pillar [5]arene by creating a quaternary carbon atom through a nucleophilic addition reaction of pillar [4]arene [1] endo-[P4-(OH)BPO] as protein binding pocket mimetic. FBDD is an approach to develop potent smallmolecule compounds starting from fragments binding weakly to target proteins 9,10 . Owing to its advantages such as saving experimental cost, offering diverse hits, and exhibiting multiple ways of binding, FBDD has been playing important roles in target-based drug discovery. In FBDD, fragment ligands are usually small molecules with low complexity in chemical structure, and H-bonds play an important role in stabilizing the fragment-pocket complexes [9][10] . For example, among the 462 unique fragment-pocket complexes investigated by Giordanetto, Shaw and co-workers, 92% of the fragments have at least one hydrogen bond formed with a protein, a structural water molecule, or a metal atom 10 . Binding of fragments to endo-[P4-(OH)BPO]. Recently, several biomimetic pockets with polar binding sites in their hydrophobic pockets have been disclosed [11][12][13][14][15][16][17][18] . Impressively, thermodynamics of interactions between biomimetic pockets and guests in water has been studied systematically by Jiang and coworkers [16][17][18] , which is highly valuable as the recognizations of ligands by protein binding pockets occur in aqueous environment in living systems. However, it is challenging to assess the contribution to pocketligand binding a nity "solely" from hydrogen bonding only through studies on the complexation in water. The reasons include: a) water molecules intend to bind the inner polar site of a synthetic receptor, and b) water molecules compete with a host in binding with a polar guest, weakening H-bond interaction between the host and guest [16][17][18] . In order to better understand both big picture and details of molecular recognition, investigation of pocket-fragment complexation in non-polar environment should be indispensably complimentary to the studies in aqueous solutions. Therefore, we initiated a thermodynamic study on the fragment-pocket binding abilities and selectivities of endo-[P4-(OH)BPO] with respect to various guest compounds in a non-polar solvent, CDCl 3 . In selecting guests for the study, ideal guest compounds resembling "fragment ligands" in FBDD are those not only geometrically complementary to the tubular binding pocket, but, more importantly, bearing H-bond acceptor(s) or donor(s) or both so that such a host-guest interaction could mimic a protein-ligand interaction event staged synergistically by both hydrogen bonding and hydrophobic interaction 9,10 . We selected an array of small-molecule fragment compounds for a "fragment library" which includes alkyl amines, alcohols, aldehydes, acids, esters, small heterocycles, and simple small molecules possessing bilogically important functional groups (Fig. 3). 1 H NMR experiments were performed on all of the host-guest pairs at 1∶1 ratio in CDCl 3 (Supplementary Information). The shifts of the proton signals of the complexed guests relative to the free species were used to evaluate the binding events. The binding constants (K a ) and binding free energies were determined by the 1 H NMR titration method (Table 1) 24 . Generally, the energy of a single Hbond is somewhat between a van der Waals interaction and a fully covalent or ionic bond, but it varies depending on the nature of the donor and acceptor atoms, their geometries, and surrounding environments 25,26 . Thus, the "fragment-pocket" interaction of [P4-(OH)BPO] with a guest was expected to be affected by the number of H-bonds, angles of the hydrogen bond(s), nature of the donor and acceptor atoms, and the hydrophobic interaction between the inner surface of endo-[P4-(OH)BPO] and the guest 25,26 . In order to explore the correlations of the binding strength (K a ) and properties of guest fragments, molecular descriptors including the logarithm of the octanol:water partition coe cient (log P), dipole moment (μ), molecular volume (V), surface area (S), and asphericity Ω a ) were collected 16,27 .   Table 1.
With the formation of fragment-pocket complexes con rmed, we wondered whether the host was in its endo conformation where the inward-pointing OH group forms a H-bond with a guest. Unambiguous evidence of endo conformation of [P4-BPO(1-OH)] in the complex was obtained from the X-ray diffraction analysis. The single crystal structure of the complex 13⊂endo-[P4-BPO(1-OH)] (Fig. 2c) revealed that 13 is encapsulated in the host pocket with its alkyl chain in the hydrophobic tubular region of the host cavity, and, unmistakably, with its carbonyl oxygen H-bonded to the inwardly pointing OH group in the deep cavity of the host, with the CO···HO distance of 1.941 Å, perfectly in the range of a typical H-bond length 35 Table 1). Besides H-bonding, hydrophobic interaction also plays a signi cant role in stabilizing the fragment-pocket complexes in which the hydrophobic fragments of the ligands are energetically favored at the interface of the host pocket 38,39 Thus, the binding mode of an amide fragment in a "fragment-pocket" complex is determined not only by the strength of amide carbonyl-hydroxyl (CO···HO) H-bond, but also by the hydrophobic interaction between the inner cavity of the host and the guest chain 38,39 . This was well re ected in complexes 21⊂endo-[P4-(OH)BPO] and 24⊂endo-[P4-(OH)BPO] where alkyl chains of guests 21 and 24 were trapped in the host pocket albeit they were connected to different points of amide functional group (either to the amino N or the carbonyl C atoms, respectively), as shown by the single crystal X-ray structures ( Fig. 2e and 2f). The hydrophobic interaction between the predominantly hydrophobic inner surface of the pocket and the alkyl chain of the fragment determined the binding modes of these two complexes. As 21 was more polar (higher μ and lower log P value) than 24 (  Fig. S21, ) and the crystal structure of 25⊂endo-[P4-(OH)BPO] (Fig. 2g) indicated that the amine subgroup of 25 was left outside of the binding pocket, possibly owing to the difference in charge density and polarity between the N and C atoms associated with the C=O (Mulliken charges calculated to be -0.518556e and -0.417205e for the N and C atoms, respectively) (Supplementary Information). As a consequence, the solvation interaction between the solvent-exposed N-butyl chain and the "nonpolar" solvent (  (Fig. 2h). Among "screened" heterocyclic fragments, imidazole 29 formed fragment-pocket complex with a higher bonding constant (Table 1), which implied that 29 might use both of its H-bond acceptor (=N) and donor (-NH) in the interaction with the pocket.
Sulfoxide, oxirane, nitrile and nitro group frequently appear in pharmaceuticals [43][44][45][46][47] . We therefore included fragments dimethyl sulfoxide (DMSO) (35), 1-nitropropane (36), acetonitrile (37) and 2butyloxirane (38) in our exploration of fragment-pocket interaction study. DMSO molecule is dipolar aprotic with a strong polar sulfoxide group and two hydrophobic methyl moieties 43,44 , which facilitate the formation of a stable complex 35⊂endo-[P4-BPO(1-OH)] (Fig. 2f). With a nitro group that forms H-bond with a -OH group 45  We were delighted to see that the binding constants of designed bifunctional "lead molecules" 39 and 40 were enhanced by more than 13 and 20 folds, respectively, compared to their monofunctional analog 23 (Table 1). We were delighted to see that 40 achieved the highest binding strength among all of the fragments listed in Table 1  OH)] than that of 39 could be partially explained by the higher diploe moment and lower log P of the former than the latter (Table 1). In the 1 H NMR spectra of both complexes ( Supplementary Fig. S45  Complexation through "induced-t". It is generally believed that the binding of a ligand to a conformationally free protein is mechanistically via "conformational selection", whereby a ligand selectively binds to a form of the protein pocket, or via "induced t", whereby a ligand binds to a predominantly free conformation of a protein, followed by a conformational change of the protein to form a preferred protein-ligand complex 49,50 .  Supplementary Fig. S4), most probably owing to interconversion between the endoand exo-conformational isomers in a rate faster than the NMR timescale. We therefore deduced that formation the fragment-pocket complexes G⊂endo-[P4-(OH)BPO] (G = 1-40) could be an "induced t" process where a fragment guest passing through the host tube clutches the freely moving OH through H-bond interaction, and, as a consequence, freezes conformationally free [P4-(OH)BPO] into an endo-complex. Based on this assumption, we conjectured that an exo-[P4-(OH)BPO] conformational isomer could be also "frozen" out by non-polar fragment guest geometrically complementary. As expected, exo-[P4-(OH)BPO] conformational isomer was "frozen" out by solvent-induced crystallization in hexane, and was con rmed by 1 H and 2D NOSEY NMR spectra in CDCl 3 (Supplementary Fig. S47 and S175) and unambiguously single crystal X-ray structure (Fig. 6) in which the phenyl group linked to the quaternary carbon atom lands on the inner side of the tubular core, leaving the hydroxyl group pointing outside, a hexane molecule is imprisoned in the tubular cavity through solely hydrophobic interaction (Fig. 6).

Conclusion
In summary, we have fabricated a pillar [5]arene-derived molecular tube [P4-(OH)BPO]. With an inwardly pointing hydrogen bond donor (OH) in the deep cavity and a hydrogen bond acceptor (C=O) on the inner surface, plus a hydrophobic tubular inner wall, the endo-conformational isomer, endo-[P4-BPO(1-OH)], renders itself a perfect arti cial protein binding pocket. On the basis of the study on the binding behavior of various FBDD "fragment ligands" inside the "pocket" of endo-[P4-(OH)BPO], "lead molecules" were rationally designed with their binding constants greatly enhanced compared to the screening-generated "hit moleclues". The further work in this direction could be useful in FBDD and arti cial protein-ligand binding models to facilitate our understanding of complex molecular interactions in living systems.

Methods
General methods. Unless otherwise noted, all commercial reagents and solvents were used without puri cation. Separation by ash column chromatography was performed on silica gel (200-300 mesh). 1 H and 13 C NMR spectra were recorded on Bruker AVANCE NEO 400 NMR spectrometer in CDCl 3 at 298K.
Atmospheric pressure chemical ionization (APCI) mass spectra were recorded on a Thermo Scienti c Q Exactive Focus (UltiMate 3000 HPLC) mass spectrometer. Single crystal X-ray diffraction data were collected at 100 K on a Rigaku-Oxford Diffraction SuperNova dual source diffractometer (Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation). The data collected were processed by CrysAlisPro. The structures were solved by direct methods using Olex2 software, and the non-hydrogen atoms were re ned anisotropically with SHELXL-2018 using a full-matrix least-squares procedure based on F 2 . The weighted R factor, wR and goodness-of-t S values were obtained based on F 2 .The hydrogen atom positions were xed geometrically at the calculated distances and allowed to ride on their parent atoms.
Crystallographic data for the structures disclosed in this paper have been deposited at the Cambridge Crystallographic Data Center (CCDC). Calculations. Quantum chemical calculations were performed using Gaussian 16 32 . Fragment library compounds and designed "drug molecules" were calculated by RMO62X 6-311G (d,p) level of theory, and vibrational frequency calculations have be carried out to verify that all optimized geometries are true minima. Molecular volume (V) and surface area (S) of guest moleculars were calculated using Marching Tetrahedron algorithm via Multiwfn program 29 . Dipol momnetvalues were calculated using Gaussian 09 at the RB3LYP/6-31++G level of calcualtion.
Asphericity(Ω a ) was de ned as I A , I B and I C are the principal moments of inertia of a molecule 28   Structures of fragment library compounds and the designed "lead molecules".

Supplementary Files
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